Orange transgenic fluorescent ornamental fish

ABSTRACT

The present invention relates to transgenic fluorescent orange ornamental fish, as well as methods of making such fish by in vitro fertilization techniques. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing.

The present application claims the priority benefit of U.S. provisionalapplication No. 61/723,018, filed Nov. 6, 2012, which is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to transgenic fish, particularly orangefluorescent transgenic fish.

2. Description of Related Art

Transgenic technology involves the transfer of a foreign gene into ahost organism enabling the host to acquire a new and inheritable trait.Transgenic technology has many potential applications. For example, itcan be used to introduce a transgene into a fish in order to create newvarieties of fish. There are many ways of introducing a foreign geneinto fish, including: microinjection (e.g., Zhu et al., 1985; Du et al.,1992), electroporation (Powers et al., 1992), sperm-mediated genetransfer (Khoo et al., 1992; Sin et al., 1993), gene bombardment or genegun (Zelenin et al., 1991), liposome-mediated gene transfer (Szelei etal., 1994), and the direct injection of DNA into muscle tissue (Xu etal., 1999). The first transgenic fish report was published by Zhu etal., (1985) using a chimeric gene construct consisting of a mousemetallothionein gene promoter and a human growth hormone gene. Most ofthe early transgenic fish studies have concentrated on growth hormonegene transfer with an aim of generating fast growing fish. While amajority of early attempts used heterologous growth hormone genes andpromoters and failed to produce these fish (e.g. Chourrout et al., 1986;Penman et al., 1990; Brem et al., 1988; Gross et al., 1992), enhancedgrowth of transgenic fish has been demonstrated in several fish speciesincluding Atlantic salmon, several species of Pacific salmons, and loach(e.g. Du et al., 1992; Delvin et al., 1994, 1995; Tsai et al., 1995).

The zebrafish, Danio rerio, is a model organism for vertebratedevelopmental biology. As an experimental model, the zebrafish offersseveral major advantages such as easy availability of eggs and embryos,tissue clarity throughout embryogenesis, external development, shortgeneration time and easy maintenance of both the adult and the young.Transgenic zebrafish have been used as an experimental tool in zebrafishdevelopmental biology. However, for the ornamental fish industry thedark striped pigmentation of the adult zebrafish does not aid in theefficient display of the various colors that are currently available onthe market. More recently, Lamason et al. (2005) in their report showedthat the Golden zebrafish carry a recessive mutation in the slc24a5gene, a putative cation exchanger, and have diminished number, size, anddensity of melanosomes, which are the pigmented organelles of themelanocytes and hence are lightly pigmented as compared to the wild typezebrafish. The availability of such fish having modified pigmentationfor transgenesis with fluorescent proteins would result in betterproducts for the ornamental fish industry due to better visualization ofthe various colors.

Many fluorescent proteins are known in the art and have been used toinvestigate various cellular processes, including fluorescent proteinsexhibiting various green, red, yellow, blue, or purple colors. Althoughtransgenic experiments involving fluorescent proteins have provided newmarkers and reporters for transgenesis, progress in the field ofdeveloping and producing ornamental fish that express such proteins hasbeen limited.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention concerns making transgenicfluorescent fish and providing such fish to the ornamental fishindustry.

In some embodiments, transgenic fish or methods of making transgenicfish are provided. In certain aspects, the transgenic fish are fertile,transgenic, fluorescent fish. In a particular embodiment, the fish foruse with the disclosed constructs and methods is the Golden zebrafish.Zebrafish skin color is determined by pigment cells in their skin, whichcontain pigment granules called melanosomes (black or brown color),xanthosomes (yellow color), erythrosomes (orange or red color), oriridosomes (iridescent colors, including white color). The number, size,and density of the pigment granules per pigment cell influence the colorof the fish skin. Golden zebrafish have diminished number, size, anddensity of melanosomes and hence have lighter skin when compared to thewild type zebrafish. Golden zebrafish have a mutation in slc24a5 gene,rendering the fish skin lighter or less pigmented (Lamason et al.,2005).

In certain specific embodiments there are provided transgenicfluorescent zebrafish comprising specific transgenic integration events,referred to herein as transformation events. These fish are ofparticular interest because, for example, they embody an aestheticallypleasing orange color. Transgenic fish comprising these specifictransgenic events may be homozygous or heterozygous (including, forexample, hemizygous) for the transformation event. Homozygous fish bredwith fish lacking a transformation event will in nearly all casesproduce 100% heterozygous offspring. Eggs, sperm, and embryos comprisingthese specific transgenic events are also included as part of theinvention.

In one such embodiment regarding a specific transgenic integrationevent, an orange transgenic zebrafish is provided comprisingchromosomally integrated transgenes, wherein the zebrafish comprises the“Orange zebrafish 2 transformation event,” sperm comprising the Orangezebrafish 2 transformation event having been deposited as ECACCaccession no. 12103002. In certain aspects, such a transgenic zebrafishis a fertile, transgenic zebrafish. In more specific aspects, such azebrafish is a transgenic Golden zebrafish. Such a transgenic zebrafishmay be homozygous or heterozygous (including, for example, hemizygous)for the integrated expression cassette.

Also disclosed are methods of providing a zebrafish comprising theOrange zebrafish 2 transformation event to the ornamental fish market.In some embodiments, the method comprises obtaining a transgeniczebrafish comprising chromosomally integrated transgenes, wherein thezebrafish comprises the “Orange zebrafish 2 transformation event,” spermcomprising the Orange zebrafish 2 transformation event having beendeposited as ECACC accession no. 12103002, and distributing the fish tothe ornamental fish market. Such fish may be distributed by a grower toa commercial distributor, or such fish may be distributed by a grower ora commercial distributor to a retailer such as, for example, amulti-product retailer having an ornamental fish department.

In some aspects, methods of producing a transgenic zebrafish areprovided comprising: (a) obtaining a zebrafish that exhibitsfluorescence and comprises one or more chromosomally integratedtransgenes or expression cassettes, wherein the zebrafish comprises the“Orange zebrafish 2 transformation event,” sperm comprising the Orangezebrafish 2 transformation event having been deposited as ECACCaccession no. 12103002; and (b) breeding the obtained zebrafish with asecond zebrafish to provide a transgenic zebrafish comprising the Orangezebrafish 2 transformation event. The second zebrafish may be atransgenic or non-transgenic zebrafish.

In further embodiments, also provided are methods of producing atransgenic organism, the method comprising using sperm comprising theOrange zebrafish 2 transformation, such sperm having been deposited asECACC accession no. 12103002, to produce transgenic offspring. Suchoffspring may be, for example, a zebrafish, a species of the Daniogenus, a fish species or genus related to zebrafish, or another fishspecies or genus. In some aspects, the fish may be produced using invitro fertilization techniques known in the art or described herein.

As used in this specification, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Any embodiment of any of the present methods, kits, and compositions mayconsist of or consist essentially of—rather thancomprise/include/contain/have—the described features and/or steps. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION Transgenic Fish

In some aspects, the invention regards transgenic fish. Methods ofmaking transgenic fish are described in, for example, U.S. Pat. Nos.7,135,613; 7,700,825; 7,834,239, each of which is incorporated byreference in its entirety.

It is preferred that fish belonging to species and varieties of fish ofcommercial value, particularly commercial value within the ornamentalfish industry, be used. Such fish include but are not limited tocatfish, zebrafish, medaka, carp, tilapia, goldfish, tetras, barbs,sharks (family Cyprinidae), angelfish, loach, koi, glassfish, catfish,discus, eel, tetra, goby, gourami, guppy, Xiphophorus, hatchet fish,Molly fish, or pangasius. A particular fish for use in the context ofthe invention is zebrafish, Danio rerio. Zebrafish are increasinglypopular ornamental animals and would be of added commercial value invarious colors. Zebrafish embryos are easily accessible and nearlytransparent. A fish that is of particular use with the disclosedconstructs and methods is the Golden Zebrafish. Zebrafish skin color isdetermined by pigment cells in their skin, which contain pigmentgranules called melanosomes. The number, size, and density of themelanosomes per pigment cell influence the color of the fish skin.Golden zebrafish have diminished number, size, and density ofmelanosomes and hence have lighter skin when compared to the wild typezebrafish. Golden zebrafish have a mutation in the slc24a5 gene, whichcodes for a putative cation exchanger localized to intracellularmembrane, thus rendering the fish skin lighter or less pigmented(Lamason et al., 2005).

Fertilization From Frozen Sperm

Fish sperm freezing methods are well-known in the art; see, e.g., Walkerand Streisinger (1983) and Draper and Moens (2007), both of which areincorporated herein by reference in their entireties. To obtaintransgenic fish disclosed herein, frozen zebrafish sperm may be used tofertilize eggs, as described in Draper and Moens (2007).

Eggs are collected as described in Draper and Moens (2007). Briefly, twofemales are placed in tricaine solution at 16 mg/100 mL water. Aftergill movement has slowed, one of the fish is removed and rinsed inwater. The fish is placed on a paper towel to dry briefly and thentransferred to a small plastic dish. With slightly damp fingers, onefinger is placed on the dorsal side of the fish. The eggs are removed bygently pressing on the ventral side of the fish, starting just behindthe pectoral fins and moving toward the tail.

The eggs from the female zebrafish are squeezed into a 35 mm plasticPetri dish. The sperm are thawed at 33° C. in a water bath for 8-10 sec.70 μl room temperature Hanks solution is added to the vial and mixed.The eggs are then immediately added to the vial and gently mixed. Thesperm and eggs are activated by adding 750 μl of fish water and mixing.The mixture is incubated for 5 minutes at room temperature. The dish isthen filled with fish water and incubated at 28° C. After 2-3 hours,fertile embryos are transferred to small dishes where they are furthercultured.

Parichy and Johnson, 2001, which is incorporated by reference in itsentirety, provides additional examples regarding in vitro fertilization.

The invention further encompasses progeny of a transgenic fishcontaining the Orange zebrafish 2 integration event, as well as suchtransgenic fish derived from a transgenic fish egg, sperm cell, embryo,or other cell containing a genomically integrated transgenic construct.“Progeny,” as the term is used herein, can result from breeding twotransgenic fish of the invention, or from breeding a first transgenicfish of the invention to a second fish that is not a transgenic fish ofthe invention. In the latter case, the second fish can, for example, bea wild-type fish, a specialized strain of fish, a mutant fish, oranother transgenic fish. The hybrid progeny of these matings have thebenefits of the transgene for fluorescence combined with the benefitsderived from these other lineages.

The simplest way to identify fish containing the Orange zebrafish 2transformation event is by visual inspection, as the fish in questionwould be orange colored and immediately distinguishable fromnon-transgenic fish.

EXAMPLES

Certain embodiments of the invention are further described withreference to the following examples. These examples are intended to bemerely illustrative of the invention and are not intended to limit orrestrict the scope of the present invention in any way and should not beconstrued as providing conditions, parameters, reagents, or startingmaterials that must be utilized exclusively in order to practice the artof the present invention.

Example 1 Orange Transgenic Zebrafish

Transgenic fish exhibiting an orange color are provided. The specifictransgenic events embodied in these fish (comprising the ZsYellow 1gene) are designated Orange zebrafish 2. Sperm from these fish may beused to fertilize zebrafish eggs and thereby breed transgenic zebrafishthat comprise these specific transgenic integration events. Sperm fromthis line was deposited at the European Collection of Cell Cultures(ECACC) as “Orange zebrafish 2” (the deposit was designated as accessionno. 12103002).

The fluorescent transgenic fish have use as ornamental fish in themarket. Stably expressing transgenic lines can be developed by breedinga transgenic individual with a wild-type fish, mutant fish, or anothertransgenic fish. The desired transgenic fish can be distinguished fromnon-transgenic fish by observing the fish in white light, sunlight,ultraviolet light, blue light, or any other useful lighting conditionthat allows visualization of the orange color of the transgenic fish.

The fluorescent transgenic fish should also be valuable in the marketfor scientific research tools because they can be used for embryonicstudies such as tracing cell lineage and cell migration. Additionally,these fish can be used to mark cells in genetic mosaic experiments andin fish cancer models.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinvention as defined by the appended claims.

References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 7,135,613-   U.S. Pat. No. 7,700,825-   U.S. Pat. No. 7,834,239-   Brem et al., Aquaculture, 68:209-219, 1988.-   Chourrout et al., Aquaculture, 51:143-150, 1986.-   Delvin et al., Nature, 371:209-210, 1994.-   Draper and Moens, In: The Zebrafish Book, 5^(th) Ed.; Eugene,    University of Oregon Press, 2007.-   Du et al., Bio/Technology, 10:176-181, 1992.-   Gross et al., Aquaculature, 103:253-273, 1992.-   Khoo et al., Aquaculture, 107:1-19, 1992.-   Lamason et al., Science, 310(5755):1782-1786, 2005.-   Penman et al., Aquaculture, 85:35-50, 1990.-   Sin et al., Aquaculature, 117:57-69, 1993.-   Szelei et al., Transgenic Res., 3:116-119, 1994.-   Tsai et al., Can. J. Fish Aquat. Sci., 52:776-787, 1995.-   Walker and Streisinger, Genetics 103: 125-136, 1983.-   Xu et al., DNA Cell Biol., 18, 85-95, 1999.-   Zelenin et al., FEBS Lett., 287(1-2):118-120, 1991.-   Zhu et al., Z. Angew. Ichthyol., 1:31-34, 1985.

What is claimed is:
 1. A transgenic zebrafish or progeny thereofcomprising chromosomally integrated transgenes, wherein the zebrafish orprogeny thereof comprises the transformation event having been depositedas ECACC accession no.
 12103002. 2. The transgenic zebrafish of claim 1,further defined as a fertile, transgenic zebrafish.
 3. The transgeniczebrafish of claim 1, further defined as a transgenic Golden zebrafish.4. The transgenic zebrafish of claim 1, wherein the fish is homozygousfor the integrated expression cassette.
 5. The transgenic zebrafish ofclaim 1, wherein the fish is heterozygous for the integrated expressioncassette.
 6. A method of providing a transgenic fish to the ornamentalfish market, comprising obtaining a transgenic zebrafish or a progenythereof in accordance with any of claim 1, and distributing the fish tothe ornamental fish market.
 7. The method of claim 6, wherein the fishare distributed by a grower to a commercial distributor.
 8. The methodof claim 6, wherein the fish are distributed by a grower or a commercialdistributor to a retailer.
 9. The method of claim 8, wherein theretailer is a multi-product retailer having an ornamental fishdepartment.
 10. A method of producing a transgenic fish comprising: (a)obtaining a zebrafish that exhibits fluorescence and comprises achromosomally integrated expression cassette encoding a fluorescentprotein, wherein the zebrafish comprises transformation event havingbeen deposited as ECACC accession no. 12103002; and (b) breeding theobtained zebrafish with a second fish to provide a transgenic fishcomprising the transformation event.
 11. The method of claim 10, whereinthe second fish is a non-transgenic fish.
 12. A method of producing atransgenic organism, the method comprising using sperm comprising thetransformation event having been deposited as ECACC accession no.12103002, to produce transgenic offspring.
 13. A progeny of a transgeniczebrafish of claim 1 that comprises a chromosomally integratedexpression cassette encoding a fluorescent protein, wherein thezebrafish and progeny exhibit fluorescence and comprise thetransformation event having been deposited as ECACC accession no.12103002.
 14. The progeny fish of claim 13, further defined as afertile, transgenic zebrafish.
 15. The progeny fish of claim 13, furtherdefined as a transgenic Golden zebrafish.
 16. The progeny fish of claim13, wherein the fish is homozygous for the integrated expressioncassette.
 17. The progeny fish of claim 13, wherein the fish isheterozygous for the integrated expression cassette.
 18. A method ofproviding a transgenic fish to the ornamental fish market, comprisingobtaining a progeny fish in accordance with claim 13, and distributingthe fish to the ornamental fish market.
 19. The method of claim 18,wherein the fish are distributed by a grower to a commercialdistributor.
 20. The method of claim 18, wherein the fish aredistributed by a grower or a commercial distributor to a retailer. 21.The method of claim 20, wherein the retailer is a multi-product retailerhaving an ornamental fish department.
 22. A method of producing atransgenic fish comprising: (a) obtaining a transgenic fish inaccordance with claim 13; and (b) breeding the obtained fish with asecond fish to provide a transgenic fish comprising the transformationevent.
 23. The method of claim 22, wherein the second fish is anon-transgenic fish.